The present invention relates to methods for detection of shikimic acid and more particularly to methods capable of detecting shikimic acid in plant material.
Shikimic acid is an important intermediate in the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan from D-glucose.
The herbicide glyphosate, N-(phosphonomethyl) glycine, inhibits the biosynthesis of aromatic amino acids which ultimately causes accumulation of shikimic acid in plants. Glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, a key enzyme in the aromatic amino acid biosynthetic pathway. Glyphosate initially causes accumulation of shikimate-3-phosphate, the substrate of EPSP synthase, which is then hydrolyzed in the plant to shikimate. Detection of shikimic acid in plants can be used to determine whether a plant has been exposed to glyphosate and can also be used to determine whether plants are resistant to this herbicide.
Gaitonde and Gordon, J. Biol. Chem., vol. 230, no. 1, p. 1043-1050 discloses a method for quantifying shikimic acid wherein a solution of shikimic acid is oxidized by periodic acid, sodium hydroxide is added to form a yellow chromophore, and glycine is added to stabilize the color. The optical density of the solution is then measured within ten minutes of adding sodium hydroxide to the solution.
Millican, Methods in Enzymology, vol. 17A, p. 352-353, 1970 discloses a method of detecting shikimic acid wherein a solution containing shikimic acid is oxidized with periodate, and then is treated with arsenite. Thiobarbituric acid is added and the solution takes on a red color. The solution is then extracted with cyclohexanone. The clear upper cyclohexanone phase contains the red color and the optical density of the cyclohexanone phase is then determined.
Singh and Shaner, Weed Technology, vol. 12, p. 527-530, 1998 discloses a method of detecting shikimic acid in plant tissue wherein a test sample containing shikimic acid was oxidized with periodic acid. The sample was then mixed with sodium hydroxide, glycine was added and the optical density was read.
Siehl, in Herbicide Activity: Toxicology, Biochemistry and Molecular Biology, R. M. Roc et al., editors, IOS Press, 1997, p. 37-65, discloses an assay method for the enzyme DAHP wherein the enzyme is incubated in the presence of its substrates phosphoenolpyruvate, and erythrose-4-phosphate. The reaction is stopped by addition of periodic acid. Sodium sulfite is later added to reduce excess periodate. Thiobarbituric acid is then added, followed in ten minutes by dimethylsulfoxide. Absorbance at 549 nm is then determined.
The present invention provides a method of detecting shikimic acid in an aqueous solution comprising the steps of a) oxidizing shikimic acid with periodic acid or a reagent comprising periodate and periodic acid (periodic acid and the reagent are referred to collectively herein as periodate); b) adding a strong base to generate a chromophore; c) adding sulfite to stabilize the chromophore; d) detecting the presence of the chromophore; and optionally e) quantifying the amount of the chromophore. The presence of the chromophore signals the presence of shikimic acid in the aqueous solution The amount of chromophore correlates directly with the amount of shikimic acid in the aqueous solution. In a preferred embodiment of the present invention, steps b and c are performed at the same time with a mixture comprising sulfite and a strong base so that reduction of excess periodate and generation of a chromophore take place at the same time.
Applicants have discovered that the use of periodic acid and periodate provides a more rapid reaction time with shikimic acid than periodic acid alone, especially when the oxidation is carried out at temperatures above ambient room temperature.
Applicants have additionally found that use of sulfite to reduce excess periodic acid and periodate greatly improves the stability of the chromophore generated by oxidation of shikimic acid followed by addition of strong base.
Applicants"" method can also be used to detect shikimic acid in tissues, preferably plant tissue. Thus, another aspect of the invention provides a method for detection of shikimic acid in a test sample comprising the steps of (a) treating the test sample with periodic acid or a reagent comprising periodate and periodic acid; (b) adding a strong base to the test sample to generate a chromophore; (c) stabilizing the chromophore with sulfite; (d) detecting the presence of the chromophore, and optionally (e) quantifying the amount of the chromophore. Preferably, steps b and c are performed at the same time with a mixture comprising sulfite and a strong base so that reduction of excess periodate and generation of a chromophore take place at the same time.
Unlike some prior methods of detecting shikimic acid, Applicant""s methods can be used directly with tissue extracts. Suitable tissue extracts can be produced without grinding or prolonged reflux of the plant material. Further, unlike some other prior methods, Applicants"" method provides a more stable chromophore such that the detection of the chromophore need not occur as soon as possible after quenching the periodic acid oxidation. The applicability of the method of the present invention to direct use with tissue samples, the ability to delay detection of the chromophore for up to at least one hour, and optional quantification of the chromophore produced make the present invention well suited for high throughput screening of tissue samples.